Increasing number of regional conflicts associating civil and military human damages in poorly accessible places raises the problem of short and medium-term survival of injured persons and of the secondary impact of aftereffects. Among usual damages in these circumstances, hemorrhagic shock and rhabdomyolysis (large destruction of muscle cells) are among the most frequent and serious. Beyond immediate survival, it is critical to prevent the resulting development of an acute renal failure (ARF), which is determinant for the prognosis of these patients. Several solutions have been proposed to reduce the immediate mortality of hemorrhagic shock. However, their success remained relative since most of the time their efficiency relies on secondary intensive care intervention. Administration of a single dose of estrogens at the time of first care is one of these solutions. Experimental data and their simplicity of use are promising. However, physiopathologic and therapeutic information remain too scarce to consider their use at short term in human. Moreover, potential protective renal impact has not been really investigated whereas it largely conditions the later prognostic. In the case of traumatic rhabdomyolysis, the immediate survival can be improved by electrolytic protective measures and use of withers. However, degradation products released by muscle cells frequently induce an acute tubule-interstitial nephropathy leading to acute renal failure. Treatment of such an acute renal failure is most of time incompatible in the case of isolated patients or large natural disaster. Moreover, it also worsens the prognosis in the context of multiple traumas. Treatments intended to limit its renal impact, such as estrogen administration, have not been investigated to our knowledge. Our preliminary works, based on a model of resuscitated hemorrhagic shock in mice, indicate that administration of a single dose of estradiol not only improve survival of animals, whatever the genre, but also reduce the intensity of the resulting acute kidney injury in female mice (not yet studied in male) and prevents long-term development of renal fibrosis. More recently, using a model of rhabdomyolyses induced by intra-muscular glycerol injection in mice, we were also able to observe a decrease in ARF-related mortality in estrogen-impregnated mice. The main goal of this project is to estimate the feasibility and the benefits in term of survival and renal protection of a single administration of estrogen (estradiol, Premarin, estetrol) at the time of initial injury during either resuscitated hemorrhagic shock or glycerol-induced rhabdomyolysis (the two experimental models already we used in preliminary works). The end-points of the project will be: 1) survival after hemorrhagic shock (with or without preliminary resuscitation) or after induction of rhabdomyolysis; 2) development of acute kidney injury, intensity, duration including evaluation of tissue damages and functional impact; 3) determination of the most efficient and well tolerated form of estrogen, including determination of its optimal dose. Expected results: the main expected result is to show that administration of a single dose of an estrogen is able to improve survival and to prevent, or at least to attenuate the resulting acute renal failure during hemorrhagic shock or rhabdomyolysis. It is expected that this beneficial effect will be observed whatever the gender. After that, we will determine which of the 3 form of estrogen used in the study is the most efficient and exhibits the highest tolerance. Taken together, these results and the availability of the different pharmacologic forms of estrogen should provide the rational for a quick translational to human therapy in the field of war and disaster medicine. That project may end up with an interventional randomized study (double blind treatment, single dose) for preventing renal impact of traumatic hemorrhagic shock or rhabdomyolysis.

Chronic kidney disease (CKD), a major socioeconomic worldwide public health burden, is characterized by a progressive decline in renal function to end stage renal disease that can occur irrespective of the cause of the renal damage once a critical number of nephrons has been lost. Understanding the physiopathology of CKD progression is therefore a prerequisite for the development of efficient preventive strategies. We have significantly contributed to the elucidation of the molecular mechanisms involved in CKD progression. By combining an experimental model of nephron reduction (Nx) with genomic and molecular approaches, we identified in EGFR (EGF receptor) the critical player of CKD as well as one of the most promising therapeutic targets of CKD progression. However, its chronic pharmacological inhibition cannot be proposed because of the severe adverse side effects. EGFR is activated by a multitude of ligands whose biological relevance has not been completely elucidated. The nature of the ligand may elicit distinct cellular responses. Our data suggest that, among these ligands, TGF-a and EGF seems to play major antinomic/divergent roles in CKD progression. More importantly, we confirmed that these results are relevant to humans since the increase of TGF-a and the decrease of EGF predict CKD progression in CKD patients. Our working hypothesis is that the duality of nature of the ligand (TGF-a vs EGF) determines the fate of kidney towards compensation or deterioration after an initial injury. These two ligands might induce specific EGFR posttranslational modifications that might feed into ligand-specific distinct genetic networks. The aim of this project is decipher the molecular mechanisms that accounts for the divergent role of EGF and TGF-a and to identify crucial specific modulators. We will combine in vivo and in vitro models, candidate and unbiased approaches with high throughput pharmacological screening. In particular, we will: • characterize the cellular events triggered by EGF and TGF-a in renal epithelial cells. We will first determine the impact of Egf and Tgfa inactivation in different CKD mouse models. Then, we will focus on the cellular events known to participate to CKD progression: cell proliferation, cell polarity partial epithelial-mesenchymal transition (EMT). • identify the genetic networks that account for the divergent role of EGF and TGF-a. We will characterize the differential expression of transcriptome in Egf-/- and Tgfa-/- mice after Nx. Then, we will map the chromatin changes that are responsible for these transcriptional effects. • determine the ligand-specific receptor modifications and interactions in kidney of mice injected with the two ligands, using mass spectrometry. To study the spatial and temporal networks of EGFR partners, we will use an in vitro BioID screening. Candidates will be then validated in cell culture and in Egf-/- and Tgfa-/- CKD mouse models. In parallel, we will try to understand the mechanisms by which Lcn2 regulates EGFR trafficking. • screen chemical libraries for drugs susceptible to selectively impact on EGF or TGF-a signaling. Biosensors able to predict TGF-a and EGF function will be used to “sense” the pharmacological effects. The most appropriate candidates will be finally validated in Nx mice. This research project is based on a multidisciplinary approach that involves cell biology, genetics transcriptomics, proteomics and pharmacological screening to improve our knowledge of the complex pathogeneses of CKD progression. The results of this research proposal should provide a novel knowledge on EGFR and the signalling pathways that control CKD progression and, by consequent, to the design of more appropriate therapeutic strategies.

Coordination of nutrient sensing and signal transduction is essential for achieving metabolic homeostasis. Misalignments of metabolism and signalling underline pathologies including metabolic syndrome and diabetes, having major socio-economic impact. It is important to understand molecular mechanisms implicated to propose novel treatment strategies. Class III phosphoinositide 3-kinase (PI3K-III) functions in all eukaryotes showing remarkable evolutionary conservation. Its activity is critical for nutrient uptake and metabolism through control of endocytic trafficking, autophagy and lysosomal function. In our recent work, we have revealed a novel crosstalk between PI3K-III and a key nutrient sensing component - insulin signalling. We discovered a novel retro-control loop in which insulin activates PI3K-III activity selective for the endocytosis, having major effect on insulin receptor trafficking and signal transduction. Functionally, it is reflected in striking phenotype of hepatic PI3K-III mutants which are presented with defective glucose metabolism. Importantly, we have discovered that acute hepatic downregulation of PI3K-III alleviates metabolic syndrome in models of insulin resistance and diabetes suggesting that PI3K-III might be a therapeutic target in metabolic conditions. Despite recent progress, our understanding of the PI3K-III regulation in vivo and its implication in human pathology are sketchy. The overreaching aim of this research proposal is to reveal the molecular mechanisms of NUTRIent SENSing and metabolism as a function of PI3K-III (NUTRISENSPIK). In this program, we will develop and apply approaches to discover novel molecular mechanisms of hepatic PI3K-III regulation at different levels and will elucidate the metabolic activities downstream of PI3K-III. Importantly, we will also characterise the status of hepatic PI3K-III signalling in liver disease both in animal models and in human patient samples. The later will include the conditions of metabolic stress associated with nutritional challenge. Those analyses will provide a crucial information on PI3K-III signalling in common liver pathologies. In the translational perspective, this work might validate the PI3K-III signalling and its novel targets as diagnostic or prognostic markers and will rationalise testing of PI3K-III modulators in the conditions of metabolic disalignment. When completed, NUTRISENSPIK research program will fill the gap in the knowledge on the role of PI3K-III signalling in metabolic homeostasis, will offer the tools to evaluate the activity of PI3K-III and will provide the evidence on the implication of PI3K-III signalling in human liver pathophysiology. Altogether, those will advance our fundamental understanding of the mechanisms of nutrient sensing in physiology and pathology.

The pancreatic islets are key micro-organs of the endocrine system that allow the maintenance of glycemic control through hormone secretion, mainly beta cell-derived insulin. Increasing evidence suggests a role of the immune system to finely tune metabolic homeostasis. As such, macrophages residing inside pancreatic islets have recently emerged as an important component that may support islet integrity and regulate insulin secretion. Islet macrophages have a surprising activated phenotype even at steady-state and a certain level of islet inflammation seems required for homeostasis. Our preliminary data suggest that macrophage activation may be primed by a specific metabolic reprogramming orchestrated by cystine flux, while being tightly controlled by the TGFb pathway. I propose that this cystine-TGFb balance may shape islet macrophage phenotype and subsequently, impact on beta cell function. My project aims at deciphering the metabolic demands and molecular mechanisms underlying such activation in islets in both females and males at steady-state and in the pathological context of obesity and type 2 diabetes. Learning from islet macrophage endogenous and intrinsic signaling pathways may benefit to the understanding of endocrine pathologies.

Primary antibody deficiencies (PADs) are the most common primary immunodeficiency (PID) in humans. We showed recently that hyper-activation of PI3K signalling is a frequent cause for PADs. Autosomal dominant mutations in PIK3CD encoding p110d, the catalytic subunit of PI3K, specifically expressed in lymphocytes, and PIK3R1, encoding p85a, the regulatory subunit of PI3K, ubiquitously expressed, have been shown to result in Activated PI3K-d Syndromes (APDS1 and APDS2, respectively). APDS patients presented often, already in early childhood, with recurrent sino-pulmonary infections. Benign lymphoproliferation are common and some APDS patients developed B cell lymphomas. Predominant immunological features are antibody deficiency, most frequently hyper-IgM (HIGM) syndrome, increased transitional B cells, decreased naive B and naive T cells and increased frequency of effector T cells especially CD8+CD57+ T cells, so-called senescent T cells. APDS patient-derived T cells have increased level of phosphorylated AKT and ribosomal protein S6, and are sensitive to activation-induced cell death. In contrast, an immunodeficient patient lacking the p85a subunit of PI3K was described as suffering from a profound defect in B cell development and Ig production. Together, these studies suggest that PI3K signal levels should be strictly regulated in lymphocytes for an optimal immune response. The research proposal aims to further elucidate the mechanisms responsible for the immunodeficiency in APDS1 and APDS2 patients. We hypothesize that hyper-active PI3K activity might affect naive and effector lymphocyte subsets differently in their function. Thus, we will quantify phosphorylation of AKT and S6 in various lymphocyte subsets (e.g. transitional, naive and memory B cells, naive, memory and senescent CD8 T cells) present in the blood of healthy donors and APDS1 and APDS2 patients. Furthermore we will characterize the impact of hyper-activated PI3K signalling on gene expression by gene expression profiling of patients’ and controls’ T lymphocytes. The antibody deficiency present in APDS patients could be explained by an intrinsic B cell defect associated with disturbed survival and/or differentiation of naïve B cells into plasma cells. To investigate this hypothesis we will analyze in vitro differentiation of B cells into plasma blasts. Besides an intrinsic B cell defect, extrinsic defects could occur, such as impaired T follicular helper (TFH) cell function. Thus the frequency and function of TFH-like cells in the blood of APDS patients and controls will be examined. In addition, we will characterize the phenotype and function of T regulatory cells in APDS patients. The clinical and immunological features observed in APDS patients are variable from one patient to another, even in the same family, suggesting the influence of other factors (environmental or modifier genes). To identify genetic components for the phenotypic variability we will perform RNA-Sequence analysis with whole blood from several APDS1 and APDS2 patients to identify single nucleotide variation (SNV) in « modifier » gene(s) (collaboration with Sergey Nejentsev, Cambridge, UK). Our work might thus result in the identification of biomarkers and functional tests to evaluate and monitor treatment efficiency with potential drugs, e.g. mTOR- and p110d-inhibitors. We presume that genetic defects associated with hyper-activation or misbalanced PI3K/AKT/mTOR signalling could be also responsible for other PADs presenting with similar clinical and immunological phenotypes. Thus we attempt to identify and characterize these novel molecular causes for PIDs patients. Together, from this project we expect to better understand PI3K function in lymphocytes, the physiopathogeny of APDS and the identification of biomarkers for diagnosis and monitoring of treatment efficiency. It also aims to define new gene defects responsible for APDS-like patients.